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• W2031145183 abstract "A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an amino-terminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon. A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an amino-terminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon. Clarification of the basic structure of desmoplakin has been followed by the identification of many related proteins, such as BPAG1,1 plectin, envoplakin, and periplakin (1Green K.J. Parry D.A.D. Steinert P.M. Virata M.L.A. Wagner R.M. Angst B.D. Nilles L.A. J. Biol. Chem... 1990; 265: 2603-2612Google Scholar, 2Tanaka T. Parry D.A.D. Klaus-Kovtun V. Steinert P.M. Stanley J. J. Biol. Chem... 1991; 266: 12555-12559Google Scholar, 3Sawamura D. Li K. Chu M.L. Uitto J. J. Biol. Chem... 1991; 266: 17784-17790Google Scholar, 4Wiche G. Becker B. Luber K. Weitzer G. Castanon M.J. Hauptmann R. Stratowa C. Stewart M. J. Cell Biol... 1991; 114: 83-99Google Scholar, 5McLean W.H.I. Pulkkinen L. Smith F.J.D. Rugg E.L. Lane E.B. Bullrich F. Burgeson R.E. Amano S. Hudson D.L. Owaribe K. McGrath J.A. McMillan J.R. Eady R.A.J. Leigh I.M. Christiano A.M. Uitto J. Genes Dev... 1996; 10: 1724-1735Google Scholar, 6Ruhrberg C. Hajibagheri M.A.N. Simon M. Dooly T.P. Watt F.M. J. Cell Biol... 1996; 134: 715-729Google Scholar, 7Ruhrberg C. Hajibagheri M.A.N. Parry D.A.D. Watt F.M. J. Cell Biol... 1997; 139: 1835-1849Google Scholar). These proteins form a family known as the “plakin family” (8Uitto J. Pulkkinen L. Smith F.J.D. McLean W.H.I. Exp. Dermatol... 1996; 5: 237-246Google Scholar). Almost all members of this family have a common structure, with predicted globular amino-terminal and COOH-terminal domains that are separated by a central rod domain. Some homologous domain structures have been identified in both globular domains of many plakins, while the central domain is rich in heptad repeats and is believed to form a parallel α-helical coiled-coil structure with a dimerization partner (9Green K.J. Virata M.L.A. Elgart G.W. Stanley J. Parry D.A.D. Int. J. Biol. Macromol... 1992; 14: 145-153Google Scholar). As suggested by this model, it has been demonstrated that desmoplakin I can form homodimersin vitro (10O'Keefe E.J. Erickson H.P. Bennett V. J. Biol. Chem... 1989; 264: 8310-8318Google Scholar). Early investigations revealed that the COOH-terminal domains of plakins are involved in binding to intermediate filaments (11Stappenbeck T.S. Bornslaeger E.A. Corcoran C.M. Luu H.H. Virata M.L.A. Green K.J. J. Cell Biol... 1993; 123: 691-705Google Scholar, 12Wiche G. Gromov D. Donovan A. Castanon M.J. Fuchs E. J. Cell Biol... 1993; 121: 607-619Google Scholar, 13Nikolic B. Nulty E.M. Mir B. Wiche G. J. Cell Biol... 1996; 134: 1455-1467Google Scholar). The amino-terminal domains of desmoplakin and of BPAG1 are believed to bind to desmosomes or hemidesmosomes. Furthermore, some splicing variants of plectin and BPAG1 have actin- or microtubule-binding domains at their amino termini, and it has been proposed that these domains form cross-links between microfilaments and/or microtubules and intermediate filaments (14Yang Y. Dowling J., Yu, Q.C. Kouklis P. Cleveland D.W. Fuchs E. Cell.. 1996; 86: 655-665Google Scholar, 15Yang Y. Bauer C. Strasser G. Wollman R. Julien J.P. Fuchs E. Cell.. 1998; 98: 229-238Google Scholar, 16Fuchs P. Zoerer M. Rezniczek G.A. Spazierer D. Oehler S. Castanon M.J. Hauptmann R. Wiche G. Hum. Mol. Genet... 1999; 8: 2461-2472Google Scholar). Studies of a few inheritable diseases that appear to involve plectin or desmoplakin and of a BPAG1 null mouse have shown that each plakin plays a critical role in the tissue integrity in specific tissues (5McLean W.H.I. Pulkkinen L. Smith F.J.D. Rugg E.L. Lane E.B. Bullrich F. Burgeson R.E. Amano S. Hudson D.L. Owaribe K. McGrath J.A. McMillan J.R. Eady R.A.J. Leigh I.M. Christiano A.M. Uitto J. Genes Dev... 1996; 10: 1724-1735Google Scholar, 17Gache Y. Chavanas S. Lacour J.P. Wiche G. Owaribe K. Meneguzzi G. Ortonne J.P. J. Clin. Invest... 1996; 97: 2289-2298Google Scholar, 18Armstrong D.K.B. Mckenna K.E. Purkis P.E. Green K.J. Eady R.A.J. Leigh I.M. Hughes A.E. Hum. Mol. Genet... 1999; 8: 143-148Google Scholar, 19Guo L. Degenstein L. Dowling J., Yu, Q.C. Wollmann R. Perman B. Fuchs E. Cell.. 1995; 81: 233-243Google Scholar). Moreover, it seems likely that, in many autoimmune blistering diseases, plakins, located in the epidermis, might be target antigens, and these plakins are used for markers of specific diseases (2Tanaka T. Parry D.A.D. Klaus-Kovtun V. Steinert P.M. Stanley J. J. Biol. Chem... 1991; 266: 12555-12559Google Scholar, 20Oursler J.R. Labib R.S. Ariss-Abdo L. Burke T. O'Keefe E.J. Anhalt G.J. J. Clin. Invest... 1992; 89: 1775-1782Google Scholar, 21Kim S.C. Kwon Y.D. Lee I.J. Chang S.N. Lee T.G. J. Invest. Dermatol... 1997; 109: 365-369Google Scholar, 22Proby C. Fujii Y. Owaribe K. Nishikawa T. Amagai M. J. Invest. Dermatol... 1999; 112: 153-156Google Scholar). However, their pathological roles remain to be clarified. Several years ago we described an individual with a subepidermal blistering disease that resembled bullous pemphigoid both clinically and pathologically (23Fujiwara S. Shinkai H. Takayasu S. Owaribe K. Tsukita S. Kageshita T. J. Dermatol... 1992; 19: 610-613Google Scholar). Immunoblot analysis revealed that the patient's serum did not react with the 230-kDa (BPAG1) and 180-kDa bullous pemphigoid antigens, whereas it did recognize a 450-kDa epidermal polypeptide. This polypeptide was expressed in human keratinocytes and in some transformed cell lines that included HeLa, KB, and A431 cells (24Fujiwara S. Kohno K. Iwamatsu A. Shinkai H. Dermatology.. 1994; 189: 120-122Google Scholar). In a preliminary study, to determine the molecular structure of this antigen, we screened a human keratinocyte cDNA library with the patient's serum. We isolated two kinds of cDNA: one encoding a protein that was strongly homologous to rat plectin and another encoding a protein, with partial homology to plectin, which appeared to be a novel and previously unidentified protein (25Fujiwara S. Kohno K. Iwamatsu A. Naito I. Shinkai H. J. Invest. Dermatol... 1996; 106: 1125-1130Google Scholar). We report here the cloning of the cDNA and determination of the entire structure of the novel protein, which we compare with other members of the plakin family. We also demonstrate the tissue localization of this protein, as determined by Northern blotting and immunostaining. In our previous study, it was unclear whether the epitope that was recognized by our patient's serum was a sequence that is present within plectin itself. In this study, therefore, we also identified the major epitope of the protein. A cDNA library prepared from HeLa cells (CLONTECH) was screened with the inserts of clones pE450-C and pE450-D, which had been isolated by extension cloning, using the same library, after the immunoscreening described in a previous report (25Fujiwara S. Kohno K. Iwamatsu A. Naito I. Shinkai H. J. Invest. Dermatol... 1996; 106: 1125-1130Google Scholar). For isolation of the 3′ end repeats, we constructed a cDNA library from the poly(A)+ RNA of HeLa cells. cDNA was synthesized with a cDNA synthesis kit (Life Technologies, Inc.). We modified the procedure provided with the kit by adding trehalose to the reaction for first-strand synthesis (26Carninci P. Nishiyama Y. Westover A. Itoh M. Nagaoka S. Sasaki N. Okazaki Y. Muramatsu M. Hayashizaki Y. Proc. Natl. Acad. Sci. U. S. A... 1998; 95: 520-524Google Scholar) and, as specific primers for first-strand synthesis, we used 5′-CCAGACACAACAAGTATGCC-3′ for clone Ep115 and 5′-TAGCGCTTGACCGAGTCCATC-3′ for clone Ep 4. The cDNAs were ligated to an EcoRI adapter and then inserted into the EcoRI site of pUC 18 (Amersham Pharmacia Biotech) for construction of plasmid libraries. Each library was screened with the specific probe. To confirm the size of 3′ end repetitive region and cover a small gap of the message, genomic polymerase chain reaction (PCR) was performed using primers 5′-TCGAGAAGCAGGAAACCA-3′ and 5′-CCATATGACACATAGACGAC-3′. To obtain some 5′-upstream cDNA sequences, we performed 5′-RACE (rapid amplification of cDNA ends) using the RACE System (Life Technologies) and total RNA from HeLa cells or the Marathon-Ready cDNAs System with adaptor-ligated double-stranded cDNA prepared from poly(A)+ RNA from HeLa cells (CLONTECH). The products of RACE were cloned into the pGEM-T Easy vector (Promega). Each cDNA clone and genomic clone of interest was sequenced by a double-strand strategy with an automatic DNA sequencer (Applied Biosystems). Internal primers were constructed for analysis of internal sequences. We searched various protein data bases, including Swiss-Prot and PIR, using the BLAST routine available from the National Center for Biotechnology Information (Bethesda, MD). Furthermore, we also made comparisons using the COMPARE and DOTPLOT programs available from the University of Wisconsin Genetics Computer Group (Madison, WI). We also used the MATCH routine to identify regions of sequence homology among proteins. Determinations of plausible secondary structure were made using several predictive techniques and results are presented here only in a preliminary form. Short regions of heptad substructure were delineated by hand rather than by direct computer analysis since the former method facilitated location of discontinuities in heptad phasing. Potential interchain ionic interactions between charged residues at positions 2d′-1g, 1g′-2e, 2a′-1g, 1g′-2a, 1e′-1d, and 1d′-1e were considered as a function of relative chain stagger and chain polarity (27McLachlan A.D. Stewart M. J. Mol. Biol... 1975; 98: 293-304Google Scholar, 28Parry D.A.D. Crewther W.G. Fraser R.D.B. MacRae T.P. J. Mol. Biol... 1977; 113: 449-454Google Scholar). The notation 2d′-1g means that the residue at position d of the second heptad of chain n′ interacts with that in position g of the first heptad of chain n. Overlapping peptides were synthesized on derivatized cellulose membranes with Fmoc (9-fluorenylmethoxycarbonyl) amino acids according to the protocol from the manufacturer of the system (Auto spot robot ASP222; ABIMED Analysen-Technik GmbH, Langenfeld, Germany) (29Frank R. Overwin H. Methods Mol. Biol... 1996; 66: 49-169Google Scholar, 30Appel J.R. Pinilla C. Niman H. Houghten R.A. J. Immunol... 1990; 144: 976-983Google Scholar). The ASP222 software program was used to generate the amino acid sequences of decapeptides and the spotting schedule for each cycle of addition of an amino acid. Peptides spanning amino acid residues 2807–3337 of epiplakin were synthesized on cellulose membranes as a series of decapeptides with an overlap of eight amino acids. This region includes one of five highly conserved COOH-terminal repeats. The first cDNA clone that we obtained by immunoscreening encoded a part of this domain. To refine the definition of the epitope within the sequence LVPAKDQPGRQEKMSIYQAMWKGVLRPGT, we synthesized a series of decapeptides with an overlap of nine amino acids were synthesized on cellulose membranes. Enzyme immunoassays of the peptides on the cellulose membranes were performed with our patient's serum, alkaline phosphatase-conjugated antibodies against human IgG, and 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. All reagents were purchased from Bio-Rad. The target peptides, namely peptide A (MSIYQAMWKGVLC), peptide B (TKGFFDPNTHENC), and peptide C (VKRYLEGTSCIAGVLVP), were synthesized by a solid-phase method (31Hudson D. J. Org. Chem... 1988; 53: 617-624Google Scholar). Peptide chains were elongated using an automated peptide synthesizer (model 9050; Millipore/Biosearch, Marlborough, MA) according to the standard operating programs. Peptides were purified by high-performance liquid chromatography on a reverse-phase column (Delta PakC18; Waters) with a Multisolvent Delivery System (600E; Waters). Peptides were eluted with a linear gradient from water to acetonitrile in 0.1% trifluoroacetic acid. The molecular mass and amino acid sequence of each purified peptide were confirmed by determinations of the matrix-assisted laser desorption ionization time of flight mass spectrometer (Kompact MALDI II; Kratos-Shimadzu, Tokyo, Japan) and a protein sequencer (491CLC; Applied Biosystems). Each synthetic peptide (2 mg) was conjugated to bovine serum albumin (BSA; 10 mg; Sigma) usingN-(4-maleimidobutyryloxy)succinimide (2 mg; Dojin, Kumamoto, Japan) according to the manufacturer's protocol. The ELISA was performed with microplates and BSA-conjugated peptides A, B, and C. Wells of microtiter plates (Nunc A/S, Kamstrupvej, Roskilde, Denmark) were coated with 50 ng of synthetic peptide that had been conjugated to BSA (0.5 μg/ml in PBS) with incubation at 4 °C overnight. After incubation with PBS that contained 0.05% Tween 20 (PBS-T) for 1 h at room temperature to block nonspecific binding, samples of serum (diluted 1:200 or serial 2-fold dilutions in PBS-T that contained 1% BSA) were added to the wells. After a 1-h incubation with shaking at room temperature, the wells were washed three times with PBS-T, and 100 μl of a solution of alkaline phosphatase-conjugated antibodies against human IgG antibody (Bio-Rad) that had been diluted to 1:1000 in PBS-T were added. After incubation for 1 h with shaking at room temperature, the wells were washed three times with PBS-T and twice with PBS. After a 1-h incubation with the substrate solution at room temperature, absorbance (405 nm) was determined with a SOFTMAX system (Molecular Devices Co., Sunnyvale, CA). The absorbance obtained with each synthetic peptide was corrected by subtraction of the value due to reaction of the antibodies with BSA by itself. Epidermal extracts were obtained as described previously (23Fujiwara S. Shinkai H. Takayasu S. Owaribe K. Tsukita S. Kageshita T. J. Dermatol... 1992; 19: 610-613Google Scholar). After addition of dithiothreitol at a final concentration of 0.01 m, the solubilized proteins were separated by electrophoresis on an SDS-polyacrylamide gel (5% polyacrylamide) and transferred to an Immobilon™ polyvinylidene difluoride membrane (Millipore), which was probed with the original patient's serum or polyclonal antibodies against the synthetic peptide (see below) before or after incubation with the synthetic peptide. The cDNA that encoded a specific region of epiplakin was amplified by PCR with specific primers and subcloned in pGEX 4T-1 as an EcoRI fragment. The expression of the GST recombinant protein in Escherichia coli JM 109 was induced by 1 mm isopropyl-1-thio-β-d-galactopyranoside at 32 °C in LB medium supplemented with 50 μg/ml ampicillin. After culture for 3 h with isopropyl-1-thio-β-d-galactopyranoside, bacterial cells were harvested and lysed by sonication in PBS, with subsequent centrifugation at 15,000 × g for 10 min. Supernatants were incubated with 40 μl/ml glutathione-Sepharose (Amersham Pharmacia Biotech) for 30 min at 20 °C. The glutathione-Sepharose beads were washed with elution buffer that contained 15 mmglutathione to elute the GST recombinant proteins. The amino acid sequence of the protein was confirmed with the automated sequencer after Western blotting of proteins on Immobilon™ membranes. Antibodies against a recombinant protein were raised in rats. The purified GST recombinant protein was mixed with an equal volume of Freund's complete or incomplete adjuvant. Initial subcutaneous injections contained 100 μg of recombinant protein (plus complete Freund's adjuvant) per rat. Two booster injections, each consisting of the same amount of protein (plus Freund's incomplete adjuvant) were given 1 and 2 weeks after the initial injection. The rats were bled 3 weeks after the second booster injection. The inguinal lymph nodes were dissected out for production of monoclonal antibodies. Purified peptide A (MSIYQAMWKGVLC) was conjugated with keyhole limpet hemocyanin using N-(4-maleimidobutyryloxy)succinimide and rabbits were immunized with the conjugate plus Freund's complete adjuvant. Antibodies (peptide A-specific antibodies) were purified by immunoaffinity gel chromatography on a peptide-immobilized column with absorption in 1 m NaCl (pH 7.5) and subsequent elution with 0.17 m Gly-HCl (pH 2.3) at room temperature. An RNA Master Blot (PT3004-1) was purchased from CLONTECH and Northern dot blotting was performed according to the protocol fromCLONTECH. The product of PCR obtained with 5′-GGCGCCAGGACCTGCTGA-3′ and 5′-CACCTTGCTGAGCCGCTCCT-3′ as primers and Ep12 as template was labeled with 32P and used as the specific probe for epiplakin mRNA. The product of PCR obtained with 5′-CCACACCACGGTGGACGA-3′ and 5′-ACTCAGCAGCTGCCTCTG-3′ as primers and pE 450-B as template was used as the specific probe for plectin mRNA (25Fujiwara S. Kohno K. Iwamatsu A. Naito I. Shinkai H. J. Invest. Dermatol... 1996; 106: 1125-1130Google Scholar). Ubiquitin cDNA was used as a control probe. Human tissues were obtained at biopsy or autopsy, embedded into OCT compound (Sakura Fine Technical Co., Ltd., Tokyo, Japan) and frozen in liquid nitrogen. Frozen sections (5 μm) were air-dried and incubated in PBS for 5 min and then for 1 h at room temperature in PBS that contained 100-fold diluted specific antiserum or antiserum against GST. The sections were washed three times in PBS and then incubated with fluorescein isothiocyanate-conjugated (FITC-conjugated) goat antibodies against rat (for peptide-specific antiserum) or against goat (for GST-specific antiserum) IgG for 1 h at room temperature, with three subsequent washes in PBS. Sections were mounted in immersion oil (Olympus) and examined under a fluorescence microscope (UFX-DX; Nikon). We first screened the HeLa cell λgt11 library of random-primed cDNA using the 5′ end of clone pE 450-D as probe (Fig. 1). The longest clone, designated clone Ep24, was isolated, and primers from the 5′ end of clone Ep24 were used for 5′-RACE to identify five overlapping clones (clones Ep28, Ep11, Ep2, Ep79, and Ep21). The original HeLa cell cDNA library was then screened again, and clones Ep1 and Ep5 were obtained. In clone Ep5, the putative initiation codon ATG was preceded by an in-frame termination codon. For downstream cloning, we screened the HeLa cell cDNA library using the 3′ end of clone pE 450-C (Fig. 1). We isolated several almost identical clones and a clone that included TGA (clone Ep12). From these results, we postulated that the 3′ side of the message had highly homologous repeats of 1602 bp. Clones pE 450-C and pE 450-D both overlapped with parts of the repetitive region. However, the corresponding position could not be identified in this region because of the presence of five strongly homologous repeats. We constructed a cDNA library using a specific primer from the 3′- terminal region of clone Ep12. The longest clone, Ep115, was 6.3 kilobases long and it was isolated together with several shorter clones, Ep150 and Ep104. We constructed another cDNA library using a specific primer from the repetitive region of the 1.6-kilobase pair repeat and isolated a single clone, designated Ep 4. We performed genomic PCR to amplify the repetitive region and obtained a clone that was slightly more than 9 kilobase pairs in length and contained no introns. The gap of 760 bases in the message that had resulted in a gap in the cDNA was, thus, covered by genomic sequence. It appeared that an open reading frame began at the second ATG triplet, which was preceded by an in-frame TGA triplet, because the first ATG triplet was not preceded by a consensus sequence that would favor the initiation of translation (32Kozak M. Nucleic Acids Res... 1987; 15: 8125-8148Google Scholar). The total length of the ORF was 15,195 bp, and the predicted amino acid sequence (5065 amino acids) is shown in Fig.2. The theoretical relative molecular mass was calculated to be 552,467 Da and was in reasonable agreement with the molecular mass of 450 kDa determined previously by SDS-polyacrylamide gel electrophoresis. The key feature of the protein was the presence of 13 B domains of the type first identified in the COOH-terminal domain of desmoplakin (1Green K.J. Parry D.A.D. Steinert P.M. Virata M.L.A. Wagner R.M. Angst B.D. Nilles L.A. J. Biol. Chem... 1990; 265: 2603-2612Google Scholar). This feature alone clearly identified epiplakin as a member of the plakin family, which includes desmoplakin, BPAG1, and plectin. However, several unusual features were also found, as follows. The B domains themselves could be divided into two groups: one group with ∼70% identity to the B domain in desmoplakin (domains 3, 6, and 8–13; indicated by B in Fig.1), and another group with ∼45–50% identity to the B domain in desmoplakin (domains 1, 2, 4, 5, and 7; indicated by B* in Fig. 1). However, all 13 repeats were more similar to the B domain rather than to the A or C domain that were also first recognized in the COOH-terminal domain of desmoplakin (TableI and Ref. 1Green K.J. Parry D.A.D. Steinert P.M. Virata M.L.A. Wagner R.M. Angst B.D. Nilles L.A. J. Biol. Chem... 1990; 265: 2603-2612Google Scholar). We found, moreover, that the linker regions (358 residues in length) that preceded domains 9–13 and followed these five B domains were almost perfectly identical (Fig.3 A). In addition, we identified three homologous segments within each of five strongly conserved linker regions (Fig. 3 B). For example, in the linker region (residues 2391–2748), two of the segments (residues 2391–2472 and 2473–2554) were 30% identical, with particularly high identity over the last 63 residues. After a glycine-proline-rich segment, a third homologous region (residues 2613–2675), which was shorter than the other two, similarly showed homology over the same region. We predicted that these quasi-repeats should contain two or three heptad-containing segments that are separated from one another by β-turns and/or short β-strands. The total potential number of heptad-containing segments was nine, and we postulated that these segments should be grouped in two or three bundles that contain antiparallel α-helices, a motif found in the structures of many other globular proteins. We failed to identify a coiled-coil rod domain and an amino-terminal domain, both of which are characteristic features of all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence, suggesting that epiplakin probably exists in vivo as a single-chain structure. A search for other functional protein motifs did not reveal any transmembrane sequences or any vimentin- or actin-binding domains (13Nikolic B. Nulty E.M. Mir B. Wiche G. J. Cell Biol... 1996; 134: 1455-1467Google Scholar, 16Fuchs P. Zoerer M. Rezniczek G.A. Spazierer D. Oehler S. Castanon M.J. Hauptmann R. Wiche G. Hum. Mol. Genet... 1999; 8: 2461-2472Google Scholar).Table IComparison between segments of epiplakin and the COOH-terminal domains A, B and C of desmoplakinDesmoplakinEpiplakinABCDomain no.SegmentIdentities%IdentIdentities%IdentIdentities%Ident127–20464368146*52302271–44166397946*46273596–77186491226957324916–108878458348*523051232–140768398247*523061557–1732834712370573271883–205877448749*523082215–2390814612370553192749–29248347124705632103283–34588347124705632113817–39928347124705632124351–45268247123705531134885–50608347124705531All 13 domains, with the exception of domains 1, 2 and 4, consisted of 176 amino acid residues. These other three domains consisted of 178 (domain 1), 171 (domain 2), and 173 (domain 4) residues, respectively. An asterisk indicated B domains of epiplakin that are approximately 46–49% homologous to the B domain in desmoplakin. Open table in a new tab Figure 3The structure of the COOH-terminal domains of epiplakin. A, comparison of the sequences of five COOH-terminal, strongly conserved, homologous repeats. Each repeat consisted of the linker regions (358 residues in length) followed by B domains 9, 10, 11, 12, and 13 (176 residues in length). In total, 534 amino acids were almost perfectly identical. Amino acids that are identical in five repeats are shaded. Asterisk(*) indicates the different amino acids. B, comparison of the sequences of three homologous segments within one of five strongly conserved linker regions (residues 2391–2748). The sequences of three homologous segments are compared with each other. Two of these segments (residues 2391–2472 and 2473–2554) are 30% identical, with particularly homology over the last 63 residues. The third homologous region (residues 2613–2675) is shorter than the former two but, similarly, exhibits the homology over the same region. Amino acids that are identical in three segments are shaded.View Large Image Figure ViewerDownload (PPT) All 13 domains, with the exception of domains 1, 2 and 4, consisted of 176 amino acid residues. These other three domains consisted of 178 (domain 1), 171 (domain 2), and 173 (domain 4) residues, respectively. An asterisk indicated B domains of epiplakin that are approximately 46–49% homologous to the B domain in desmoplakin. To map the linear epitope in epiplakin accurately, we tested the original patient's serum, which had been used for cDNA screening, for reactivity against 264 consecutive cellulose-bound linear peptides of 10 amino acids in length with an 8-amino acid overlap in the region between amino acids residues 2807 and 3337 of epiplakin (Fig. 2). This region covers one of five strongly conserved COOH-terminal repeats and includes the sequence that was recognized by the patient's serum (25Fujiwara S. Kohno K. Iwamatsu A. Naito I. Shinkai H. J. Invest. Dermatol... 1996; 106: 1125-1130Google Scholar). The patient's serum reacted strongly with the amino acids sequence MSIYQAMWKGVL and weakly with amino acids TKGFFDPNTH. The former sequence is unique to epiplakin, but the latter is also found in plectin and is highly homologous, with the exception of glutamic acid at the last position instead of histidine (TKGFFDPNTE), to a peptide in desmoplakin. To confirm the reactivity of the serum, we performed an ELISA and found that the patient's serum reacted only with the former peptide (Fig.4 A). To refine the identification of the epitope, we exposed a series of decapeptides with nine overlapping amino acids derived from the sequence LVPAKDQPGRQEKMSIYQAMWKGVLRPGT (residues 2739–2767) to the patient's serum in another dot blot test. We detected four strongly positive spots, corresponding to sequences from KMSIYQAMWK to IYQAMWKGVL (Fig.4 B) and, thus, the major epitope recognized by the patient's serum was determined to be IYQAMWK (Fig. 4 C). Western blotting showed that the patient's serum, after absorption with the synthetic peptide MSIYQAMWKGVLC, no longer reacted with the 450-kDa epidermal autoantigen (Fig. 5,lanes 1 and 2). These data indicated that antibodies in the patient's serum recognized mainly the unique epitope in epiplakin.Figure 5Western blotting using several antisera against the 450-kDa epidermal antigen. An extract of human epidermis was subjected to immunoblotting analysis with the patient's serum before (lane 1) and after (lane 2) incubation with the synthetic peptide MSIYQAMWKGVLC, and with polyclonal antibodies against the synthetic peptide before (lane 3) and after (lane 4) incubation with the same synthetic peptide. Polyclonal antiserum (lane 5) and monoclonal antibodies (lane 7) against the GST-epiplakin fusion protein, but not the control polyclonal antiserum (l" @default.
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• W2031145183 title "Epiplakin, a Novel Member of the Plakin Family Originally Identified as a 450-kDa Human Epidermal Autoantigen" @default.
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